**2. Occurrence of antibiotic resistance in WWTPs**

a main public health concern by the World Health Organization [2, 3]. Antibiotics are most common strategy used in the treatment of bacterial infections, in addition, antibacterial metals are widely used to prevent bacterial attachment and to combat biofilms in hospital and

Antibiotics have been detected in sewage effluents, ground and surface water, sewage sludge, soil, and manure. Studies on the fate of antibiotics are motivated by two main concerns; first, antibiotics in the environment may contribute to the development of antibiotic resistant pathogens, second, the ecological consequences of antibiotic contamination that may enter in the human food chain. In a study conducted by Clarke and Smith on antibiotics in biosolids, norfloxacin, ofloxacin, ciprofloxacin, and doxycycline were measured in the sludge of a Swedish WWTP. Similar concentrations of ciprofloxacin and norfloxacin were also observed in sewage sludge samples from Switzerland. The concentration of these antibiotics was at the

compounds and concentration were measured in soil that was amended by biosolids. Some of the compounds, for instance, carbamazepine and sulfamethazine can be translocated from the

Kim and Aga [5] studied the effects of antibiotics and ARB of wastewater treatment plants (WWTPs) on ecology and human health. Their study introduced the WWTPs as a point contamination source of persistent pharmaceuticals that affect the design and operation of treatment systems, antibiotic resistance development among pathogenic bacteria, and accumulation of persistent pharmaceuticals in soil and water. Their study estimated concentrations of antibiotics in untreated municipal wastewater in the United States and showed their possible metabolites in activated sludge [5]. They concluded that the disappearance of the parent pharmaceuticals in WWTPs does not certainly mean their complete removal. The presence of pharmaceuticals in the aquatic environment has ecotoxicological effects that impact the algal community structure and shifts the food web structure of streams [5]. The potential ecological and health impacts of antibiotics in the environment were investigated using environmental risk assessment including a two-stage process; estimation of expected introductory concentration (EIC) entering the environment and predicted environmental concentration (PEC). PEC is needed if the drug has the potential to bioaccumulate in the environment [5].

Determination and characterization of pharmaceutical compounds, antibiotics in particular, has attracted attention because of their ecotoxicological effects [5–7]. Antibiotics, such as tetracycline, sulfamethoxazole, ciprofloxacin, norfloxacin, trimethoprim, and ofloxacin, are determined in high concentration in the sludge of different WWTPs. In a study directed by Martin [6] sludge from four sludge stabilization treatments including anaerobic digestion, aerobic digestion, composting and the lagoon was monitored to detect the occurrence of 22 pharmaceutically active compounds. The average concentrations of studied compounds were 179, 310 and 142 μg/kg of sludge dry matter in primary sludge, secondary sludge, and mixed sludge, respectively. Sewage sludge that is used for the land application is always treated during one or more treatment processes namely, lime stabilization, thickening, dewatering, drying, anaerobic digestion or composting processes. However, many contaminants like anti-

biotic compounds may not be removed efficiently [6].

soil into the aerial plant components by uptake mechanisms in greenhouse plants [4].

dry weight range and was constant during the treatment processes. The same

food processing settings [2].

76 Antimicrobial Resistance - A Global Threat

low mg kg−<sup>1</sup>

High concentration of antibiotics and their associated ARB and ARGs in the effluent of WWTPs enter the environment through WWTPs discharges to rivers, wastewater reuse, irrigation and amending the soil by biosolids make. Antibiotic resistance genes can persist in the environment even when there is no antibiotic pressure.

Du et al. [9] studied ARGs including *tet(X), tet*(*W*), *tet(G), sul(1)*, and *intI(1)* in the influent and effluent of different units of a municipal WWTP. The studied plant possessed the anaerobic/ anoxic/aerobic membrane bioreactors (MBR). The decrease of ARGs in anaerobic and anoxic units followed by an increase of ARGs in aerobic units and then decline of ARGs in MBR units was reported in this study [9]. Anaerobic and anoxic treatments methods were more effective than aerobic treatment methods at removing ARGs. Because microorganisms have lower bioactivity under anaerobic condition and the propagation of resistance genes are inhibited [9]. Furthermore, a significant positive correlation was observed between ARGs and 16S rDNA in the wastewater treatment process [9].

Wang et al. [10] conducted a study to explore the concentration of five tetracyclines, four sulfonamides, and six fluoroquinolones in the rhizosphere soil that was irrigated by reclaimed wastewater for a long time. The total concentration of tetracycline was in the range of 12.7– 145.2 μg kg−<sup>1</sup> while no sulfonamide was found in samples. Fluoroquinolones were randomly detected in soils and their highest total concentration was 79.2 μg kg−<sup>1</sup> . Based on the results of this study, soils that are irrigated by reclaimed wastewater accumulate antibiotics in several folds higher concentrations compared to the antibiotic concentration in the wastewater [10].

Wang and his research group studied soils of six public parks which were irrigated by the reclaimed wastewater. There was no antibiotic pressure but sulfonamide resistance genes (*sul(1)* and *sul(2)*) persisted in the soil. This result indicated that ARGs are more permanent


experimental evidence showing a relation between the acquisition of HMRGs and ARGs by

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Genetic co-selection of resistance genes occurs when in the presence of a stress, the selection of the associated resistance gene results in the persistence of additional resistance genes [20, 22]. Co-selection happens even without a straight effect of their specific stressors. Antibiotics and metals, as sources of environmental stresses, can affect bacterial antibiotic susceptibility and heavy metal resistance promotion. Regularly the presence of mobile genetic elements (MGEs) carrying multiple resistant genes results in co-selection [20, 23]. The molecular mechanisms behind the development of heavy metal resistance are almost similar to mechanisms which

There are several studies investigating the common structural and functional characteristics of antibiotic-resistance and metal-resistance systems. Antibiotics namely, chloramphenicol, ciprofloxacin, coumermycin, rifampicin, tetracycline, and trimethoprim and also metals like

In another study, low total metal levels correlate with ARG abundance in soils, implying that low metal levels may co-select for antibiotic resistance [24]. In this study, the abundance of 11 ARGs was quantified by quantitative polymerase chain reaction (qPCR) assay and compared with the metal levels in the selected soils. Copper, chromium, nickel, lead, and iron signifi-

Icgen and Yilmaz [19] conducted a research on the Kızılırmak River which receives industrial discharges to study co-occurrence of heavy metal and antibiotic resistance in bacteria. Twenty-four isolates were found resistant to both heavy metal and antibiotics. Resistance to heavy metals involving lead, tin, nickel, barium, aluminum, strontium, silver and lithium ranged from 50 to 92% and more than 50% of the isolates were resistant to cephalosporin, quinolone, sulfonamide and aminoglycoside classes of antibiotics. Therefore, the discharge of antimicrobials to surface water may result in co-selection of heavy metal- and antibioticresistant bacteria [19]. The level of heavy metal in the river varied directly with changes in industrial discharge and rainfall. The relation between heavy metal exposure level and metal-

Antibacterial properties of heavy metals may contribute to development of resistance. Antibacterial properties of nine pure metals including titanium, cobalt, nickel, copper, zinc, zirconium, molybdenum, tin, and lead have been studied using two bacterial strains, Grampositive *Staphylococcus aureus* and Gram-negative *Escherichia coli* [25]. Based on the results, the antibacterial properties of various metals were different and among the tested metals, titanium and tin did not exhibit antibacterial properties [25]. Among the nine mentioned metals, copper and zinc are common metals in WWTPs [26–28] which are in contact with ARGs

and HMRGs. Following paragraphs explain ARGs and HMRGs correlation in detail.

High concentrations of anthropogenic metal contamination in the environment can apply co-selection pressure and result in antibiotic-resistance through genetic couplings [28].

*2.1.1. Resistance mechanism acting on both metals and antibiotics*

explain antibiotic resistance like efflux, by which MGEs transfer genes [23].

As, Cu, Zn, Mn, Co, Ag, Hg, Cd, and Ni have been studied [21].

cantly correlated with the abundance of ARGs [24].

and antibiotic-resistance was not clarified.

the mechanism of co-selection [20, 21].

**Table 1.** Results of studies on ARGs concentrations (tetracycline and sulfonamide) in WWTPs.

rather than antibiotics [10]. Based on the results of a study on removal of ARB and ARGs from urban wastewater, the abundance of 16S rRNA, *intI1*, *sul(1)*, *qnrS*, *blaCTX-M*, and *blaTEM* was increased to pre-treatment amount after 3 days of storage of treated wastewater [11]. Hence, it is important to find effective processes to prevent bacterial reactivation before discharge or reuse of wastewater. **Table 1** is reporting concentrations of ARGs in samples from biosolid and effluent of different WWTPs.

#### **2.1. Effect of metal on ARGs development at WWTPs**

The world is getting progressively more industrialized and urbanized which leads to elevation of heavy metals concentrations into the environment. Human activities such as mining, waste disposal, and corrosion of metals introduce more metal contaminations into the environment [18]. Population growth and industrial development have resulted in the increase in the discharge of industrial effluents in the environment. The effluent contains antibiotics and heavy metals which can trigger antibiotic- and heavy metal- resistance. ARB and heavy metal resistant bacteria and their associated genes are a public health concern.

Municipal wastewater is a hotspot for emerging contaminants namely antibiotics, heavy metals, ARGs, and heavy metal resistance genes (HMRGs). There are bacteria like *Escherichia coli* and *Salmonella* that are resistant to multiple antibiotics and heavy metal [19]. There is some experimental evidence showing a relation between the acquisition of HMRGs and ARGs by the mechanism of co-selection [20, 21].

Genetic co-selection of resistance genes occurs when in the presence of a stress, the selection of the associated resistance gene results in the persistence of additional resistance genes [20, 22]. Co-selection happens even without a straight effect of their specific stressors. Antibiotics and metals, as sources of environmental stresses, can affect bacterial antibiotic susceptibility and heavy metal resistance promotion. Regularly the presence of mobile genetic elements (MGEs) carrying multiple resistant genes results in co-selection [20, 23]. The molecular mechanisms behind the development of heavy metal resistance are almost similar to mechanisms which explain antibiotic resistance like efflux, by which MGEs transfer genes [23].

There are several studies investigating the common structural and functional characteristics of antibiotic-resistance and metal-resistance systems. Antibiotics namely, chloramphenicol, ciprofloxacin, coumermycin, rifampicin, tetracycline, and trimethoprim and also metals like As, Cu, Zn, Mn, Co, Ag, Hg, Cd, and Ni have been studied [21].

In another study, low total metal levels correlate with ARG abundance in soils, implying that low metal levels may co-select for antibiotic resistance [24]. In this study, the abundance of 11 ARGs was quantified by quantitative polymerase chain reaction (qPCR) assay and compared with the metal levels in the selected soils. Copper, chromium, nickel, lead, and iron significantly correlated with the abundance of ARGs [24].

Icgen and Yilmaz [19] conducted a research on the Kızılırmak River which receives industrial discharges to study co-occurrence of heavy metal and antibiotic resistance in bacteria. Twenty-four isolates were found resistant to both heavy metal and antibiotics. Resistance to heavy metals involving lead, tin, nickel, barium, aluminum, strontium, silver and lithium ranged from 50 to 92% and more than 50% of the isolates were resistant to cephalosporin, quinolone, sulfonamide and aminoglycoside classes of antibiotics. Therefore, the discharge of antimicrobials to surface water may result in co-selection of heavy metal- and antibioticresistant bacteria [19]. The level of heavy metal in the river varied directly with changes in industrial discharge and rainfall. The relation between heavy metal exposure level and metaland antibiotic-resistance was not clarified.

Antibacterial properties of heavy metals may contribute to development of resistance. Antibacterial properties of nine pure metals including titanium, cobalt, nickel, copper, zinc, zirconium, molybdenum, tin, and lead have been studied using two bacterial strains, Grampositive *Staphylococcus aureus* and Gram-negative *Escherichia coli* [25]. Based on the results, the antibacterial properties of various metals were different and among the tested metals, titanium and tin did not exhibit antibacterial properties [25]. Among the nine mentioned metals, copper and zinc are common metals in WWTPs [26–28] which are in contact with ARGs and HMRGs. Following paragraphs explain ARGs and HMRGs correlation in detail.

#### *2.1.1. Resistance mechanism acting on both metals and antibiotics*

rather than antibiotics [10]. Based on the results of a study on removal of ARB and ARGs from urban wastewater, the abundance of 16S rRNA, *intI1*, *sul(1)*, *qnrS*, *blaCTX-M*, and *blaTEM* was increased to pre-treatment amount after 3 days of storage of treated wastewater [11]. Hence, it is important to find effective processes to prevent bacterial reactivation before discharge or reuse of wastewater. **Table 1** is reporting concentrations of ARGs in samples from biosolid

**Treatment Method Target Biosolid (copies/mL) Effluent (copies/mL) Reference**

1.00 × 108

3.09 × 108

1.23 × 108

2.51 × 108

3.16 × 108

9.7 × 104 8.7 × 104 1.8 × 105 5.6 × 104 2.2 × 105

9.53 × 108 3.15 × 108 6.04 × 108

2.34 × 105

6.31 × 106

5.62 × 106

–1.78 × 109 9.12 × 105

ND\*

6.31 × 103

1.58 × 104

2.5 × 102 1.6 × 102 4.4 × 102 1.6 × 101 5.5 × 103

 – 1.32 × 104 ND – 2.14 × 104

–1.58 × 106

–7.94 × 105

— [16]

–5.62 × 106

ND – 4.27 × 103 ND – 9.12 × 103 2.34× 104

–9.33 × 108

–1.29 × 109

–1.58 × 109

–2.51 × 107

–1.74 × 109

–2.51 × 109

–109

–1.05 × 106 [12]

[13]

[14]

[15]

[17]

The world is getting progressively more industrialized and urbanized which leads to elevation of heavy metals concentrations into the environment. Human activities such as mining, waste disposal, and corrosion of metals introduce more metal contaminations into the environment [18]. Population growth and industrial development have resulted in the increase in the discharge of industrial effluents in the environment. The effluent contains antibiotics and heavy metals which can trigger antibiotic- and heavy metal- resistance. ARB and heavy metal

Municipal wastewater is a hotspot for emerging contaminants namely antibiotics, heavy metals, ARGs, and heavy metal resistance genes (HMRGs). There are bacteria like *Escherichia coli* and *Salmonella* that are resistant to multiple antibiotics and heavy metal [19]. There is some

and effluent of different WWTPs.

Activated sludge *tetO, tetW,* 

Activated sludge and chlorination

78 Antimicrobial Resistance - A Global Threat

Activated sludge and chlorination *tetO*

Different WWTPs *tetW*

Conventional *tetW*

\*ND = nondetectable.

Activated sludge chlorination

and UV

*sul1*

*tetC tetA*

*tetQ tetG*

*tetQ tetW tetH tetZ*

*tetO sul1*

*tetO sul1*

**Table 1.** Results of studies on ARGs concentrations (tetracycline and sulfonamide) in WWTPs.

**2.1. Effect of metal on ARGs development at WWTPs**

resistant bacteria and their associated genes are a public health concern.

High concentrations of anthropogenic metal contamination in the environment can apply co-selection pressure and result in antibiotic-resistance through genetic couplings [28].

these genes originate from WWTPs or soils contaminated by wastewater. The class 1 integrons, specifically, are frequently associated with gene cassettes in which both HMRGs and ARGs are present and can play an important role in co-selection mechanisms [20, 25, 30].

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The effluent of WWTPs contains ARGs and HMRGs which discharges to the aquatic environment, results in the spread of ARGs and increasing the risk of gene transfer to human and animal pathogens through food chains or drinking water [1, 31]. These risks require further

However, the idea of co-selection of ARGs and HMRGs is supported by some studies, there is a lack of data determining the exposure level to antimicrobial metals on the selection of

The results of a study on the effect of Ni, Cu, Zn, Cd, and Pb on fate and distribution of ARGs, showed a positive correlation between individual ARG and HMRGs. This result implies that heavy metals act as selective stressors and lead to the co-selection mechanism between specific metal and antibiotic resistance [1]. In their study, the abundance of *sul(3), tetA, tetM, qepA,* and *qnrA* genes had a positive correlation with Cu, Zn and Hg concentration [1].

Cu or Zn are selectors of exact bacterial populations flourishing in wastewater. Zn or Cu selected for populations of *Betaproteobacteria* and *Flavobacteria* that result in multidrug resistance against carbapenems and third-generation cephalosporins [31]. Based on the review of different studies on ARGs development in WWTP, Cu, Zn are common and in high concentra-

Baker-Austin et al. [21] studied experimental evidence presenting a relation between HMRGs and ARGs. When the genes corresponding to resistant phenotypes are on the same genetic element (plasmid, transposon or integron) co-selection occurs, and this physical relation leads to the co-selection for other genes located on the same element [21]. The genetic traits contributed to mercury- resistance and antibiotic-resistance were established and showed that mercury- resistance was co-transferred with antibiotic resistance in a subset of mating between *Enterobacteriaceae* and recipients. There are two critical points that explain the importance of studying HMRG occurrence; co-selection mechanism and occurrence of resistance to antibacterial metals. Antibiotics namely, chloramphenicol, ciprofloxacin, coumermycin, rifampicin, tetracycline, and trimethoprim and also metals like As, Cu, Zn, Mn, Co, Ag, Hg, Cd, and Ni were studied by Baker-Austin et al. [21]. The present associations between metal contaminations and antibiotic resistance development implied the mechanisms of co-selection, including co-resistance and cross-resistance. This research group also reviewed the role of metals as a factor in co-selection and distribution of antibiotic resistance. As shown in **Table 2**, antibiotic resistance and metal resistance have common structural and

Based on the literature review, there are discrepancies in our current knowledge of the dominant mechanisms of co-selection for metal- and antibiotic-resistance at the population and community level and investigation of whether metals maintain a pool of horizontally

attention and consideration while WWTP effluents are reused as irrigation water [32].

**2.2. Co-occurrence of antibiotic and heavy metal resistance**

tion in the municipal wastewater [1, 26–28, 33–35].

transferable antibiotic-resistance determinants.

functional characteristics.

resistance genes.

**Figure 1.** Mechanisms of co-occurrence of metal and antibiotic resistance [2].

Co-resistance, co-regulation, and cross-resistance are mechanisms of co-selection. As shown in **Figure 1**, a close linkage between two or more diverse resistance genes is called co-resistance and is known as a mechanism of antibiotic-metal co-selection [21, 29]. It seems that coregulation can be a mechanism of antibiotic-resistance at which a number of transcriptional and translational responses to metal or antibiotic contact connected together to respond to both stresses [29]. Cross-resistance provides resistance to more than one antimicrobial agent like antibiotics and heavy metals [29].

There is a growing evidence of antibiotic resistance development derived from metal exposure. It seems that bacteria that are exposed to metals, like Cu and Zn, become resistant to metal and antibiotics simultaneously due to the metal selection of genetic elements that harbor both metal and antibiotic resistance genes [30]. There are many classes of antibiotics that can form complexes with metals and produced complexes can possess an enhanced or decayed antibiotic activity [30].

Di Cesare and his research group measured six ARGs (*tetA, sul(2), blaTEM, blaCTX-M, ermB,* and *qnrS*), two HMRGs (*czcA* and *arsB*), and the class I integron (*int1*) in different phases of three WWTPs. In their research, all the variables were classified into two groups; the first including *tetA, ermB, qnrS,* and the biotic and abiotic factors, and a second group was the genes *sul2, czcA, arsB,* and *int1*. In addition, the dynamics of *sul(2),* HMRGs, and *int1* correlated strongly. Based on this study, there is a possible relation between heavy metal contamination as well as HMRGs and spread of ARGs [20].

#### *2.1.2. Environmental impacts of antibiotic and heavy metal resistance genes*

When a bacterial community is exposed to heavy metal as a selective pressure in WWTP the potential co-selection of resistant genes is very high [20]. Studies on genes encoding for resistance against different metals and of ARGs in plasmids and integrons demonstrated that these genes originate from WWTPs or soils contaminated by wastewater. The class 1 integrons, specifically, are frequently associated with gene cassettes in which both HMRGs and ARGs are present and can play an important role in co-selection mechanisms [20, 25, 30].

The effluent of WWTPs contains ARGs and HMRGs which discharges to the aquatic environment, results in the spread of ARGs and increasing the risk of gene transfer to human and animal pathogens through food chains or drinking water [1, 31]. These risks require further attention and consideration while WWTP effluents are reused as irrigation water [32].

However, the idea of co-selection of ARGs and HMRGs is supported by some studies, there is a lack of data determining the exposure level to antimicrobial metals on the selection of resistance genes.

#### **2.2. Co-occurrence of antibiotic and heavy metal resistance**

Co-resistance, co-regulation, and cross-resistance are mechanisms of co-selection. As shown in **Figure 1**, a close linkage between two or more diverse resistance genes is called co-resistance and is known as a mechanism of antibiotic-metal co-selection [21, 29]. It seems that coregulation can be a mechanism of antibiotic-resistance at which a number of transcriptional and translational responses to metal or antibiotic contact connected together to respond to both stresses [29]. Cross-resistance provides resistance to more than one antimicrobial agent

There is a growing evidence of antibiotic resistance development derived from metal exposure. It seems that bacteria that are exposed to metals, like Cu and Zn, become resistant to metal and antibiotics simultaneously due to the metal selection of genetic elements that harbor both metal and antibiotic resistance genes [30]. There are many classes of antibiotics that can form complexes with metals and produced complexes can possess an enhanced or

Di Cesare and his research group measured six ARGs (*tetA, sul(2), blaTEM, blaCTX-M, ermB,* and *qnrS*), two HMRGs (*czcA* and *arsB*), and the class I integron (*int1*) in different phases of three WWTPs. In their research, all the variables were classified into two groups; the first including *tetA, ermB, qnrS,* and the biotic and abiotic factors, and a second group was the genes *sul2, czcA, arsB,* and *int1*. In addition, the dynamics of *sul(2),* HMRGs, and *int1* correlated strongly. Based on this study, there is a possible relation between heavy metal contamination as well as

When a bacterial community is exposed to heavy metal as a selective pressure in WWTP the potential co-selection of resistant genes is very high [20]. Studies on genes encoding for resistance against different metals and of ARGs in plasmids and integrons demonstrated that

*2.1.2. Environmental impacts of antibiotic and heavy metal resistance genes*

like antibiotics and heavy metals [29].

80 Antimicrobial Resistance - A Global Threat

**Figure 1.** Mechanisms of co-occurrence of metal and antibiotic resistance [2].

decayed antibiotic activity [30].

HMRGs and spread of ARGs [20].

The results of a study on the effect of Ni, Cu, Zn, Cd, and Pb on fate and distribution of ARGs, showed a positive correlation between individual ARG and HMRGs. This result implies that heavy metals act as selective stressors and lead to the co-selection mechanism between specific metal and antibiotic resistance [1]. In their study, the abundance of *sul(3), tetA, tetM, qepA,* and *qnrA* genes had a positive correlation with Cu, Zn and Hg concentration [1].

Cu or Zn are selectors of exact bacterial populations flourishing in wastewater. Zn or Cu selected for populations of *Betaproteobacteria* and *Flavobacteria* that result in multidrug resistance against carbapenems and third-generation cephalosporins [31]. Based on the review of different studies on ARGs development in WWTP, Cu, Zn are common and in high concentration in the municipal wastewater [1, 26–28, 33–35].

Baker-Austin et al. [21] studied experimental evidence presenting a relation between HMRGs and ARGs. When the genes corresponding to resistant phenotypes are on the same genetic element (plasmid, transposon or integron) co-selection occurs, and this physical relation leads to the co-selection for other genes located on the same element [21]. The genetic traits contributed to mercury- resistance and antibiotic-resistance were established and showed that mercury- resistance was co-transferred with antibiotic resistance in a subset of mating between *Enterobacteriaceae* and recipients. There are two critical points that explain the importance of studying HMRG occurrence; co-selection mechanism and occurrence of resistance to antibacterial metals. Antibiotics namely, chloramphenicol, ciprofloxacin, coumermycin, rifampicin, tetracycline, and trimethoprim and also metals like As, Cu, Zn, Mn, Co, Ag, Hg, Cd, and Ni were studied by Baker-Austin et al. [21]. The present associations between metal contaminations and antibiotic resistance development implied the mechanisms of co-selection, including co-resistance and cross-resistance. This research group also reviewed the role of metals as a factor in co-selection and distribution of antibiotic resistance. As shown in **Table 2**, antibiotic resistance and metal resistance have common structural and functional characteristics.

Based on the literature review, there are discrepancies in our current knowledge of the dominant mechanisms of co-selection for metal- and antibiotic-resistance at the population and community level and investigation of whether metals maintain a pool of horizontally transferable antibiotic-resistance determinants.


and amplicon detection, (d) sequencing and analysis of selected resistance-gene-specific amplicons. Based on the results of this study, bacteria of the WWTP share a mobile pool of ARGs that result in genetic exchange between clinical and WWTP bacteria. The final effluent of WWTP also contained ARB that confirms that the WWTP's final effluents are disseminating

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Recently, Zhang et al. studied both cell-free DNA and cell-associated DNA as a source for ARGs that are related to WWTPs. The cell-free DNA is extracellular DNA that can transform into other cells, and cell-associated DNA is intercellular DNA. The 0.22 μm filter intercepts intercellular DNA and extracellular DNA (filtrates contains the extracellular DNA). In this research, four ARGs (*sul(2), tet(C), blaPSE-1,* and *erm(B)*) as cell-associated and cell-free fractions were studied. The cell-associated DNA and cell-free DNA were independently extracted and ARGs copy numbers were quantified using qPCR. Based on the results of this study, cell-associated ARGs were more than ARGs fraction in the raw wastewater, however, after biological treatment, sludge settling, membrane filtration, and disinfection, cell-associated ARGs were removed considerably and cell-free ARGs removal was much lower. Therefore, the abundance ratio of cell-free ARGs to cell-associated ARGs increased. Cell-free ARGs are important pollutants from WWTPs which are potential risks to the effluent receiving environments [45]. Munir and Xagoraraki [16] quantified 18 biosolids samples from seven WWTPs using qPCR methods. The mean concentrations of *tet(W), tet(O),* and *sul(1)* in all samples of

, and 6.04 × 108

had considerably (p < 0.05) lower concentrations of ARGs compared with other biosolids treatment methods. In this study, two different sites were observed for 4 months to investigate levels of ARGs (*tet(W), tet(O),* and *sul(1)*) in soils fertilized with manure or biosolids. The concentration of ARGs was higher in manure than biosolids, but surprisingly, the results showed no notable change in the concentration of ARGs in the samples of soil, since genetic diversity and natural characteristics of background soil minimized the effect

In a recent study by D'Angelo [46] on the potential risks of the presence of antibiotic in biosolid amendments, sorption and desorption of tetracycline were indicated. Their research was on four types of amendments including biosolids, poultry manure, wood chip litter, and rice hull litter at different temperatures. The sorption and desorption equilibrium constant in municipal biosolids was 20 times higher than other amendments since the concentration of bound Al3+ and Fe3+ is higher in municipal biosolids. Results showed that the sorption of tetracycline was significantly increased after treatment with alum and treatment of amend-

The effect of treated urban wastewater irrigation on fungi diversity and soil microbial activities was studied by Alguacil and her team, in Spain. Based on this study, fungi diversity was higher in soil irrigated by fresh water, but microbial activities of soil irrigated by wastewater were much more than the soil irrigated by fresh water. Hence, wastewater not only had no negative effects on crop vitality but also developed fertility of the soil. Microbiological components are biotic factors of soil that might be altered by the increase of soil microbial biomass

, respectively. Lime-stabilized biosolids

antibiotic resistance in the environment [44].

biosolids were 9.53 × 108

of biosolids [16].

due to wastewater irrigation [47].

, 3.15 × 108

ments would effectively reduce antibiotic diffusion rates [46].

**Table 2.** Shared characteristics of antibiotic and metal resistance systems [21].
